Therapeutic bacteriophage compositions

ABSTRACT

The present invention relates to a bacteriophage composition comprising one or more (suitably two or more, or three) bacteriophages selected from Sa87, J-Sa36, Sa83, J-Sa37, or mutants thereof, use of the same for medical or non-medical applications, kits, bandage, and wound dressing comprising the same.

CROSS-REFERENCED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/237,496, filedAug. 15, 2016, which claims priority benefit to U.S. Provisional62/204,915 filed Aug. 13, 2015, which are incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions of bacteriophages, and useof the same for medical and non-medical applications.

BACKGROUND

The rising tide of human pathogens that are resistant to antibiotics hascreated an urgent need for new treatments for serious bacterialinfections. Novel approaches that circumvent traditional mechanisms ofantibiotic resistance, can be effective against biofilms, and avoiddisruption of the native gut flora are especially desirable. Thisclinical challenge has sparked renewed interest in bacteriophage (phage)therapy. Demonstration of efficacy in an animal model of infection is apreliminary step in the development of a new therapeutic agent.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a bacteriophage compositioncomprising one or more bacteriophages selected from Sa87, J-Sa36, Sa83,J-Sa37, or mutants thereof.

In another aspect there is provided a bacteriophage composition of theinvention for use as a medicament. Corresponding methods of treating adisease comprising administration of the bacteriophage composition to asubject are also provided.

In a further aspect the invention provides a bacteriophage compositionof the invention for use in treating a pulmonary bacterial infection ina subject, wherein the bacteriophage composition is administered to thesubject, and wherein the bacterial infection comprises Staphylococcusaureus.

The invention provides in one aspect use of a bacteriophage compositionof the invention in the manufacture of a medicament for use in treatinga pulmonary bacterial infection in a subject, wherein the bacteriophagecomposition is administered to the subject, and wherein the bacterialinfection comprises Staphylococcus aureus.

In another aspect there is provided a method of treating a pulmonarybacterial infection in a subject comprising administering thebacteriophage composition of the invention to the subject, wherein thebacterial infection comprises Staphylococcus aureus.

In one aspect there is provided a kit comprising: a bacteriophagecomposition according to the invention; and instructions for use ofsame.

The invention provides a method of killing Staphylococcus aureusbacteria on a surface, said method comprising applying a bacteriophagecomposition of the invention to the surface.

In another aspect there is provided use of the bacteriophage compositionor kit according to the invention for a non-medical application.

In a further aspect there is provided a bandage or wound dressingcomprising a bacteriophage composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawing, in which:

FIG. 1 shows efficacy of a phage cocktail vs. vancomycin in a S. aureuspneumonia model: statistical analysis of dosage groups. Statisticalanalysis was performed using Tukey's multiple comparisons test (GraphpadPrism 6, La Jolla, Calif.). Only groups demonstrating a significantdifference in the pairwise comparisons are shown (p<0.05). Where twogroups are compared to a third, the p value presented is the higher ofthe two pairwise comparisons. **p 0.001, *p 0.05.

DETAILED DESCRIPTION

The invention is predicated upon the surprising finding by the presentinventors that a bacteriophage composition comprising one or more(preferably at least two) bacteriophages selected from Sa87, J-Sa36,Sa83, J-Sa37, or mutants thereof, is particularly advantageous for usein both medical and non-medical applications, and particularly fortreating a pulmonary bacterial infection in a subject.

In one embodiment a bacteriophage composition comprises at least twobacteriophages selected from Sa87, J-Sa36, Sa83, J-Sa37, or mutantsthereof. In one embodiment a bacteriophage composition comprises atleast three bacteriophages selected from Sa87, J-Sa36, Sa83, J-Sa37, ormutants thereof. In another embodiment a bacteriophage compositioncomprises Sa87, J-Sa36, Sa83 or mutants thereof. Suitably abacteriophage composition may comprise Sa87, J-Sa36, and Sa83. In oneembodiment a bacteriophage composition consists essentially of Sa87,J-Sa36, and Sa83 or mutants thereof. Suitably a bacteriophagecomposition consists essentially of Sa87, J-Sa36, and Sa83.

The term “consists essentially of” as used herein means that only thebacteriophage(s) explicitly indicated are present in the bacteriophagecomposition, but that said composition may also contain a furthernon-bacteriophage constituent, such as an appropriate carrier, diluent,etc.

The term “mutant” as used herein refers to a bacteriophage differinggenetically from Sa87, J-Sa36, Sa83, or J-Sa37 by one or morenucleotides but still retaining the ability to infect and lyseStaphylococcus aureus target bacteria. In one embodiment (alternativelyor additionally) a “mutant” bacteriophage is capable of lysing the sametarget bacterial strains as Sa87, J-Sa36, Sa83, and/or J-Sa37, andfurther capable of lysing one or more additional bacterial strains. Inone embodiment a mutant may have at least 75%, 80%, 85%, 90%, 95% or 99%sequence identity across its entire genome when compared to Sa87,J-Sa36, Sa83, or J-Sa37 (suitably when compared to Sa87, J-Sa36 orSa83).

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art. Global methods align sequences from thebeginning to the end of the molecule and determine the best alignment byadding up scores of individual nucleotide pairs and by imposing gappenalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g.,Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity ofProgressive Multiple Sequence Alignment Through Sequence Weighting,Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) NucleicAcids Research 4673-4680 (1994); and iterative refinement. Non-limitingmethods include, e.g., BLAST, Match-box, see, e.g., Align-M, see, e.g.,Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment ofHighly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

The bacteriophage composition of the invention targets one or moreStaphylococcus aureus strains. In one embodiment a Staphylococcus aureusstrain targeted is a methicillin-resistant Staphylococcus aureus (MRSA).

The bacteriophages of a composition of the invention may be provided inthe form of a single therapeutic composition (preferred) or as a numberof separate compositions each comprising one or more members of thecomposition. In embodiments where the bacteriophages are provided in anumber of separate compositions, said bacteriophages may be administeredto a subject sequentially or simultaneously.

A bacteriophage for inclusion in a composition of the invention may bepropagated by any suitable method known in the art. For example one ormore bacteriophage(s) may be grown separately in host bacterial strainscapable of supporting growth of the bacteriophage. Typically, thebacteriophage will be grown in said host bacterial strain to highconcentrations, titrated and combined to form a composition of theinvention.

The amount of each bacteriophage employed (e.g. in a bacteriophagecomposition, method or use of the invention) will depend upon itsvirulence against the target bacterial species. Typically, said one ormore bacteriophage(s) may be combined to form a composition comprisingat least about 1×10⁷, 1×10⁸, 1×10⁹ or 1×10¹⁰, or 1×10¹¹ plaque formingunits (PFU) of each phage per ml of composition. Suitably, said one ormore bacteriophage(s) may be combined to form a composition comprisingat least about 1×10⁸ or 1×10⁹ PFU of each phage per ml of composition.

When selecting bacteriophages for inclusion in a composition of theinvention, the methods taught in WO 2013/164640 A1 (incorporated hereinby reference) may be used. In one embodiment said method comprises: a.providing two or more different bacteriophages, wherein each of said twoor more different bacteriophages independently retards growth of aStaphylococcus aureus species or strain; b. combining at least two ofsaid two or more different bacteriophages; and c. determining growth ofthe Staphylococcus aureus species or strain in the presence of saidcombination of two or more different bacteriophages, wherein theStaphylococcus aureus species or strain growth conditions are the sameor equivalent in steps a. and c.; d. wherein, if said combinationretards growth of the Staphylococcus aureus species or strain at leastequal to the greatest growth retardation achieved independently by anyone of said two or more different bacteriophages, the combination isaccepted as a composition of bacteriophages; and e. wherein, if saidcombination retards growth of the target bacterial species or strainless than the greatest growth retardation achieved independently by anyone of said two or more different bacteriophages, the combination isinitially rejected as a composition of bacteriophages.

The present inventors surprisingly found that by employing a method ofthe foregoing embodiment, an improved bacteriophage composition havingbacteriophages Sa87, J-Sa36 and Sa83 (and optionally mutants thereof)was obtained. Advantageously, said bacteriophage composition exhibitsimproved therapeutic efficacy against Staphylococcus aureus whencompared to conventional bacteriophage compositions and/or a compositioncomprising bacteriophages Sa87, J-Sa36, Sa83, and J-Sa37 (and optionallymutants thereof). Thus, a preferred bacteriophage composition comprisesor consists essentially of Sa87, J-Sa36 and Sa83.

In some embodiments a bacteriophage composition of the present inventionmay further comprise one or more additional bacteriophages. Said one ormore additional bacteriophages may target a Staphylococcus aureusspecies or strain, or a different bacterial target, for example selectedfrom one or more of the following genera Staphylococcus, Helicobacter,Klebsiella, Listeria, Mycobacterium, Escherichia, Meningococcus,Campylobacter, Streptococcus, Enterococcus, Shigella, Pseudomonas (e.g.Pseudomonas aeruginosa), Burkholderia, Clostridium, Legionella,Acetinobacter, Salmonella, or combinations thereof.

The one or more additional bacteriophages may be one taught in WO2009/044163 (incorporated herein by reference), a bacteriophage K and/orbacteriophage P68 described therein.

In one embodiment a bacteriophage composition comprises one or more(preferably at least two) bacteriophages selected from Sa87, J-Sa36,Sa83, J-Sa37, or mutants thereof, and a pharmaceutically acceptablecarrier, diluent, excipient or combinations thereof. Suitable carriers,diluents and/or excipients may include isotonic saline solutions, suchas phosphate-buffered saline.

A bacteriophage composition of the invention may be formulated as adisinfectant composition. The disinfectant composition may be in theform of a spray or liquid wash for a surface. The composition may be aband wash. Suitably where the composition is a formulation for topicalapplication, it may take the form of a lotion, cream, ointment, paste,gel, foam, or any other physical form as a carrier generally known fortopical administration. Such thickened topical formulations areparticularly advantageous because the formulations adhere to the area ofthe skin on which the material is placed, thus allowing a localised highconcentration of bacteriophages to be introduced to the particular areato be disinfected. For example, paraffin- and lanolin-based creams,which are particularly useful for the application of product to thenasal cavity, are generally known in the art. However, other thickeners,such as polymer thickeners, may be used. The formulations may alsocomprise one or more of the following: water, preservatives, activesurfactants, emulsifiers, anti-oxidants, or solvents.

A bacteriophage composition of the invention may be formulated fornasal, oral, parenteral, intramuscular, intravenous, subcutaneous,transdermal, ocular or aural administration. Such a bacteriophagepreparation may be used directly, stored frozen in aqueous or othersolution with an appropriate cryoprotectant (e.g. 10% sucrose), freezedried and rehydrated prior to use, or rendered stable in some otherformulation including (but not limited to) tablet, emulsion, ointment,or impregnated wound dressing or other item. For embodiments directed tothe treatment of a pulmonary bacterial infection, the bacteriophagecomposition may be formulated for pulmonary delivery via nasal or oraladministration (e.g. by aerosolisation of the bacteriophagecomposition). Thus, in one embodiment the bacteriophage composition maybe comprised in a pulmonary delivery means, such as an inhaler or arespirator.

The present invention further relates to the use of a bacteriophagecomposition herein as a medicament (e.g. for treating a Staphylococcusaureus infection). Suitably, the bacteriophage composition findsparticular use in treating a pulmonary bacterial infection, wherein thebacterial infection comprises (or consists of) Staphylococcus aureus. Abacteriophage composition comprising or consisting essentially of Sa87,J-Sa36, Sa83, or mutants thereof is particularly advantageous whentreating a Staphylococcus aureus infection (e.g. pulmonary infection).

The term “treating” as used herein is intended to encompass prophylactictreatment as well as corrective treatment (treatment of a subjectalready suffering from a disease). Preferably “treating” refers tocorrective treatment.

A use or method of the invention typically comprises administering abacteriophage composition described herein to a subject. As used herein,a “subject” is a mammal, such as a human or other animal. In oneembodiment the subject is a human subject with a Staphylococcus aureusinfection (e.g. a Staphylococcus aureus pulmonary infection).

In one embodiment a bacteriophage composition is administered to asubject at a dosage of at least about 1×10⁷ PFU of each phage or atleast about 5×10⁷ PFU of each phage. Suitably, the bacteriophagecomposition may be administered at a dosage of at least about 1×10⁸ PFUof each phage or at least about 1×10⁹ PFU of each phage. A suitabledosage range may be between about 1×10⁷ PFU of each phage to about1×10¹¹ PFU of each phage, preferably between about 5×10⁷ PFU of eachphage to about 5×10⁹ PFU of each phage.

In some embodiments the bacteriophage composition is administered atleast once, twice, three times, or four times daily. Suitably thebacteriophage composition may be administered twice daily. In oneembodiment, therefore, a dosage of at least about 5×10⁷ PFU of eachphage is administered at least once, twice, three times, or four timesdaily. In another embodiment at least about 1×10⁸ PFU of each phage isadministered at least once, twice, three times, or four times daily. Ina further embodiment at least about 1×10⁹ PFU of each phage isadministered at least once, twice, three times, or four times daily.Suitably a dosage range between about 1×10⁷ PFU of each of phage toabout 1×10¹¹ PFU of each of phage may be administered at least once,twice, three times, or four times daily. Preferably a dosage rangebetween about 5×10⁷ PFU of each of phage to about 5×10⁹ PFU of each ofphage may be administered at least once, twice, three times, or fourtimes daily.

A bacteriophage composition for use as a medicament may be administeredby any route selected on the basis of the condition to be treated. Inone embodiment the route of administration is nasal, oral, pulmonary,parenteral, intramuscular, intravenous, subcutaneous, transdermal,ocular, aural or combinations thereof. When used in the treatment of apulmonary bacterial infection, the bacteriophage composition may beadministered nasally or orally, for example via aerosolisation using anappropriate pulmonary delivery means, such as an inhaler or respirator.

In one embodiment an antibiotic (suitably a chemical antibiotic) may beadministered in combination with the bacteriophage composition of theinvention. Combinatorial administration of antibiotics andbacteriophages is taught in WO 2008/110840 and WO 2005/009451, whichteaching is incorporated herein by reference. The antibiotic may beadministered simultaneously or sequentially with the bacteriophagecomposition. Suitably, the one or more antibiotics may be administeredafter the composition such that bacteriophage replication has becomeestablished before antibiotic treatment begins. In this case, antibiotictreatment may be delayed for one or more hours or days from applicationof the one or more bacteriophages, e.g. from 1 to 2, 3, 4, 5, 6, 7, 8, 9or 10 days. Where a bacteriophage composition comprising a plurality ofbacteriophages is employed with each member of the compositionexhibiting different strain specificity, it will suffice that at least aproportion (e.g. one or more bacteriophage(s)) of the composition iscapable of targeting the bacterial infection.

Thus, in some embodiments a bacteriophage composition comprises one ormore antibiotics, such as one or more chemical antibiotics. Anantibiotic may be selected based on sensitivity of a Staphylococcusaureus species or strain to said antibiotic. Suitably the Staphylococcusaureus species or strain may be the same species or strain present in asubject to be treated. In one embodiment a Staphylococcus aureus speciesor strain is taken from a subject to be treated and tested forantibiotic sensitivity. Sensitivity may be determined by in vitrosensitivity assays known in the art.

Alternatively or additionally, an antibiotic may be selected because itis known to be active against a bacteria known to be (or thought likelyto be) present together with a Staphylococcus aureus infection to betreated (e.g. as part of a bacterial biofilm).

In one embodiment an antibiotic is one or more selected from:vancomycin, teicoplanin, penicillin, methicillin, flucloxacillin,dicloxacillin, cephalosporins (e.g. cefazolin, cephalothin, cephalexin),clindamycin, lincomycin, erythromycin, or combinations thereof. Suitablythe antibiotic may be vancomycin and/or teicoplanin.

In one embodiment a use or method of the invention comprises: a.administration of a bacteriophage composition to a subject in vivo; b.in vitro monitoring of the sensitivity of a sample of bacterial cellsfrom an infection (e.g. present in the subject) or from anotherinfection by the same strain to one or more antibiotic(s); and c.administration of said one or more antibiotic(s), when it has beenestablished that said sensitivity to said one or more antibiotic(s) hasbeen induced.

In one embodiment the antibiotic (e.g. chemical antibiotic) isadministered at a time period at which sensitivity of sampled bacteriato the antibiotic is induced by the composition. In some embodiments thetime period may be at least 12, 24 or 48 hours. In other embodiments thebacteriophage composition and the antibiotic may be administered atintervals of one day to two months apart, preferably at intervals of oneto four weeks apart, suitably at intervals of two weeks apart.

In one embodiment an antibiotic is administered at a dose of at least50, 100 or 150 mg/kg once or twice daily. Suitably an antibiotic may beadministered at a dose of 100 mg/kg once or twice daily.

In one embodiment a bacteriophage composition may be used in a method ofkilling Staphylococcus aureus bacteria on a surface, said methodcomprising applying a bacteriophage composition of the invention (e.g.formulated as a disinfectant composition) to the surface. Suitably, thesurface is a site of contamination or prospective site of contamination.

In one embodiment the surface is the skin of a mammal (e.g. a human),for example a nasal cavity. Alternatively or additionally, the surfacemay be equipment (suitably medical equipment), bedding, furniture, wallsor floors (e.g. in a clinical environment).

Suitably, a bacteriophage composition may be applied to a surface at aratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, at least 20:1,40:1, 50:1, at least 100:1 PFU of (suitably each) bacteriophage tocolony forming units (CFU) of bacteria.

The present invention also provides a kit comprising: a bacteriophagecomposition according to the invention; and instructions for use of same(e.g. in medicine). The kit may further comprise an antibiotic (e.g. achemical antibiotic) and instructions for use of same in combinationwith the bacteriophage composition.

In one embodiment the instructions provide details for dosing abacteriophage composition of the invention as described herein. In oneembodiment the instructions included in a kit of the invention are foruse of same in treating a Staphylococcus aureus infection, e.g. apulmonary infection.

The invention contemplates use of a bacteriophage composition or kit ofthe invention for non-medical applications. For example a bacteriophagecomposition or kit may be used in food hygiene, agriculture or cropprotection, and/or in environmental hygiene applications. Thus, in oneembodiment the kit comprises instructions for use of a bacteriophagecomposition in a non-medical application.

A bacteriophage composition of the invention may also be comprised in abandage or wound dressing. The wound dressing may be a pad or stickingplaster-type dressing. The bacteriophages may be applied to the wounddressing or bandage as a disinfectant formulation or topical cream,prior to applying to the wound dressing or bandage. Alternatively, thewound dressing or bandage may be soaked in a carrier containing thebacteriophages and dried to impregnate said bacteriophages within thedressing or bandage. Bacteriophages may also be adsorbed onto thesurface of the bandage or wound dressing using techniques generallyknown in the art. The advantage of this approach is that the bandage orwound dressing allows the bacteriophages to be brought into contact witha wound which may contain the bacteria. In a related aspect, the presentinvention also provides methods of inhibiting or treating bacteria byapplying a bandage or wound dressing to a subject.

The bacteriophage composition of the present invention is particularlyadvantageous for use in medicine, and shows clinical efficacy in thetreatment of Staphylococcus aureus infections. For example, it hassurprisingly been found that said bacteriophage composition isparticularly suited to treatment of Staphylococcus aureus pulmonaryinfections.

Additionally, the bacteriophage composition of the invention isefficacious against a broad spectrum of Staphylococcus aureus strains.

A combination of a bacteriophage composition and an antibiotic (e.g. achemical antibiotic) may provide an enhanced (e.g. synergistic)therapeutic showing unexpectedly improved efficacy when treating aStaphylococcus aureus infection, particularly when used in treating apulmonary Staphylococcus aureus infection.

A bacteriophage composition comprising or consisting essentially ofSa87, J-Sa36, and Sa83 (optionally further including one or more mutantsthereof) surprisingly exhibits reduced bacteriophage antagonism and/orreduces development of resistance in Staphylococcus aureus targetbacteria. Surprisingly, said bacteriophage composition exhibits reducedbacteriophage antagonism and/or reduces development of resistance inStaphylococcus aureus target bacteria when compared to a compositioncomprising or consisting essentially of Sa87, J-Sa36, Sa83, and J-Sa37(optionally further including one or more mutants thereof).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, NewYork (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, NY (1991) provide one of skill with a generaldictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects or embodiments of this disclosurewhich can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully definedby reference to the specification as a whole.

Other definitions of terms may appear throughout the specification.Before the exemplary embodiments are described in more detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “abacteriophage composition” includes a plurality of such candidate agentsand reference to “the bacteriophage” includes reference to one or morebacteriophages and equivalents thereof known to those skilled in theart, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

The invention will now be described, by way of example only, withreference to the following FIGURE and Examples.

EXAMPLES Example 1 Assembly of Bacteriophage Cocktail

In some embodiments, a cocktail of four bacteriophages Sa87, J-Sa36,J-Sa37 and Sa83, which together had broad activity against a panel ofrecent diverse S. aureus clinical isolates (unpublished data), was usedfor animal studies. In an exemplary method, phage lysates were preparedusing manufacturing hosts SPS1216 and SPS1226, which do not releaseendogenous prophage during the production cycle. Cultures were grown inbioreactors to an OD₆₀₀ 0.2 prior to phage addition. Incubation at 37°C. was continued and absorbance read at least every 60 minutes. Cultureswere harvested after bacterial lysis, and impurities separated from thephages with several filtration steps followed by chromatographic stepsthat enabled reduction of debris such as host cell proteins and hostcell DNA. After purification, lysates were additionally concentratedusing spin columns (Amicon® Ultra 15-100 kDa, Merck Millipore,Darmstadt, Germany) such that final phage titers were 1×10¹¹ PFU/mL. Atthe end of the process, buffer was exchanged and all material wasfilter-sterilized (0.22 μm filter) and stored at 2-8° C. Plaque assayswere used to titer the phage stocks (Carlson, K., In E. Kutter and A.Sulakvelidze (ed.), Bacteriophages: Biology and Application, (2005)437-494, CRC Press, Boca Raton, Fla., Hyman, P., et al., Meth. Mol.Biol. (2009) 501:175-202). The four purified phage samples were combinedsuch that each phage was present in the final cocktail at aconcentration of 2×10¹⁰ PFU/mL per phage and then additionally diluted1:10 or 1:100 to obtain the 3 dosing solutions. Final endotoxin levelswere <1000 EU/mL, which was acceptable for animal studies.

Example 2 Bacterial Strains

In a non-limiting example, Staphylococcus aureus UNT109-3 and UNT144-3used for this study are part of the UNT culture collection. Frozenstocks were inoculated into TRIPTICASE™ SOY BROTH (Triptic Soy Broth)(BBL™ Laboratories)+5% defibrinated sheep blood (TSBb) and incubated for18 hrs at 37° C. (shaking). After 18 hrs the culture was diluted 1:10into fresh TSBb and incubated for a further 5 hrs before being dilutedin fresh TSB for inoculation into the mice.

Example 3 Assessment of S. aureus Virulence in the Murine PneumoniaModel

In a non-limiting example, the sensitivity of two S. aureus clinicalisolates, UNT109-3 (NRS234, native valve endocarditis) and UNT144-3,that have been previously utilized in animal models of infection, wasevaluated vs. the individual phage and the 4-phage cocktail. In someembodiments, both strains were fully sensitive to the cocktail as wellas the individual phage Sa87, J-Sa36, and Sa83. Phages Sa87, J-Sa36, andSa83 demonstrated improved efficacy against UNT109-3 when compared tophage J-Sa37. The virulence of the two strains was then evaluated in themurine lung infection model. Female Hsd:ICR(CD-1) mice (HarlanLaboratories, Houston, Tex.) were administered 150 and 100 mg/kgcyclophosphamide on day −4 and day −1 prior to infection to render themneutropenic. Groups of 5 mice were then anaesthetized by intraperitoneal(IP) injection of 0.15 mL of a mixture of ketamine HCl (100 mg/kg bodyweight) plus xylazine (10 mg/kg body weight). Once anaesthetized, micewere infected intranasally by placing drops on the external nares andallowing inhalation of the 50 μl inoculum. Twenty-four hours afterinfection, mice were euthanized by CO₂ inhalation and lungs processedfor bacterial titers. Bacterial counts were enumerated on Brain HeartInfusion agar (Difco™ Laboratories)+0.5% activated charcoal(Sigma-Aldrich) plates for ease of recovering and detecting S. aureus(Barr, J. G., et al., J Clin. Pathol. (1987) 40:372-376). Mice that hadreceived 7.13 log₁₀ CFU of strain UNT109-3 all succumbed to theinfection prior to the 24-hour harvest, indicating unsuitable virulence.All mice infected with 6.95 log o CFU of strain UNT144-3 survived untilsampling, and exhibited mean log o CFU/lung pair titers of 6.78±0.34 and8.17±0.91 and 2 and 24 hours post-infection, respectively. These dataindicate that the virulence of the UNT144-3 strain, both in terms oflung titers and survival, is comparable to historical results obtainedfor other MRSA isolates (unpublished data). Based on these results,UNT144-3 was selected for use in the efficacy studies.

Example 4 Efficacy of the 4 Phage Cocktail in the S. aureus PneumoniaModel

In a non-limiting example, six groups of 5 mice were renderedneutropenic as described above. Once anaesthetized, an inoculum of 6.98log o CFU in 50 μL of strain UNT144-3 was delivered intranasally,resulting in mean bacterial lung titers of 7.24 log₁₀ CFU/lung pair at 2hrs, which increased to 9.18 log₁₀ CFU/lung pair at 24 hours in theuntreated control group (FIG. 1). 100 mg/kg Vancomycin was administeredas a subcutaneous injection 2 hr and 6 hr post-infection; PBS-Mg diluentwas delivered intranasally at 2 hr and 6 hr post-infection to theuntreated control group.

Three phage cocktail treatment groups were evaluated for efficacy:2×10¹⁰ PFU/mL per phage, 2×10⁹ PFU/mL per phage, and 2×10⁸ PFU/mL perphage. As described below, 50 μL doses of phage were administered atboth 2 hr and 6 hr post-infection, such that each mouse received 1×10⁹PFU of each phage, 1×10⁸ PFU of each phage, or 1×10⁷ PFU of each phageat each time point, according to its dosage group. At the time of thefirst administration of 50 μL phage, the colony counts in the lung were7.24 log₁₀ CFU/lung pair. Thus, the multiplicity of infection was ˜60,˜6 and ˜0.6 for the 3 dosage groups at the 2 hr time point when thefirst phage dose was administered.

Administration of two doses of the phage cocktail resulted in S. aureuslung titers of 6.08, 6.16 and 7.8 log¹⁰ CFU/lung pair for the 1×10⁹PFU/phage, 1×10⁸ PFU/phage, and 1×10⁷ PFU/phage treatment groups,respectively. These correspond to 1.38-3.1 log₁₀ CFU reductions comparedto the 24 hr vehicle control group and 1.08-1.16 log₁₀ CFU reductionsfor the 1×10⁹ PFU/phage and 1×10⁸ PFU/phage groups compared to bacterialtiters at 2 hrs post-infection. FIG. 1 shows a comparison of thedifferent treatment groups that demonstrated statistical significance asdetermined by ANOVA analysis (Tukey's multiple comparisons test). The1×10⁹ PFU/phage, 1×10⁸ PFU/phage, treatment groups demonstrated asignificant reduction in lung CFU vs the 24 hr non-treated control(P<0.0001 for both). Vancomycin administration (100 mg/kg SC at 2 and 6hrs) resulted in 24 hour bacterial lung titers of 5.55 log₁₀ CFU,similar to the two highest phage doses (no significant differencebetween those 3 groups was observed).

Example 5 Analysis of Colonies Resultinci from Mouse Luno Homocienatesafter Infection

In a non-limiting example, a total of 27 S. aureus colonies recoveredfrom the murine lung infection model were evaluated for sensitivity tothe individual phage components (Sa83, Sa87, J-Sa36, J-Sa37) and to the4-phage cocktail. Colonies from the 2 hr untreated cohort appearedhomogenous on the BHI-charcoal plates; thus a single colony was isolatedfrom each of 3 different mice. In some cases, both normal andtranslucent colonies were observed and therefore both colony types wereevaluated from 3 different mice per group (e.g., 24 hr untreated andvancomycin groups). Four colonies from each of the three S. aureusmorphotypes seen among phage-treated groups were also evaluated (total12): these included colonies with a typical S. aureus morphology as wellas translucent colonies. Spot tests were performed on lawns of the 27recovered colonies in order to assess phage sensitivity to the 4individual phage and the phage cocktail using 5 μL spots of the neat,10⁻¹, 10⁻², 10⁻³, and 0⁻⁴ serial phage dilutions (Carlson, K., supra,Hyman, P., supra). Testing was performed in triplicate. Susceptiblecontrol strains (SPS1216 and SPS1226) were run in parallel. Bacteriawere considered sensitive if a visible clearing of the bacterial lawn,that was attributable to productive phage infection, was observed (e.g.,consistent scoring or progression to individual plaques in serialdilutions). The 27 colonies demonstrated equivalent sensitivity toUNT144-3 for the phage cocktail as well as three individual phage Sa83,Sa87 and J-Sa36.

In this study, we demonstrated that the efficacy of a phage cocktail at1×10⁸ PFU/phage (MOI of each phage ˜6 at the time of the first dose) and1×10⁹ PFU/phage (MOI of each phage ˜60 at the time of the first dose)was comparable to vancomycin dosed at 100 mg/kg SC. Additionally, the S.aureus isolates that were re-isolated from infected mice remainedsensitive to the phage cocktail. These data provide a first step in theevaluation and development of a phage therapeutic, including initialassessment of the dosing regimen.

TABLE 1 S. aureus Neutropenic Lung Model CFU # Test Dose OR TiterAssessed Group Mice Article Route (BID) (Time) 1 5 Phage IN 1e9PFU/phage 24 hr 2 5 1e8 PFU/phage 3 5 1e7 PFU/phage 4 5 Vancomycin SC100 mg/kg 24 hr 5 5 Untreated — — 24 hr 6 5 — —  2 hr

TABLE 2 Evaluation of 4 phage cocktail vs vancomycin CFU Assessed Mean ±SD logo Dose OR Titer (Time) CFU/Lung Pair 1e9 PFU/phage BID 24 hr 6.08± 1.04 1e8 PFU/phage BID 24 hr 6.16 ± 0.89 1e7 PFU/phage BID 24 hr  7.8± 0.45 Vancomycin 24 hr 5.55 ± 1.1  Infected, untreated 24 hr 9.18 ±0.32 Infected, untreated  2 hr 7.24 ± 0.12

Example 6 Assembly of a 3 Phage Bacteriophage Composition

A number of bacteriophages (including Sa87, J-Sa36, Sa83, and J-Sa37)targeting Staphylococcus aureus are grown on permissive host strains andthen tested against a range of S. aureus strains by: spot testing onbacterial lawns, enumerative plaque assay and broth culture using aplate reader assay system. The plate reader monitors the optical densityof a broth culture containing bacteriophages with a suitable host in amulti-well plate format. This latter method allows detailed kinetics ofthe infection process to be evaluated. Bacteriophages showing goodplaque formation are selected.

Candidate bacteriophages are propagated in liquid (broth) culture andlysates prepared. Clarified lysates are purified by centrifugationthrough a sucrose cushion (27 ml of each lysate is carefullyover-layered onto 5 ml of a sterile 10% w/v sucrose ‘cushion’, in 36 mlpolypropylene tubes prior to centrifugation).

The individual bacteriophages (including Sa87, J-Sa36, Sa83, and J-Sa37)are then retested both individually at higher MOI (multiplicity ofinfection [ratio of infecting bacteriophage to bacterial host cells])and as a mixture. The results of this testing are surprising,bacteriophages Sa87, J-Sa36, Sa83, J-Sa37 produce effective reduction ofbacterial host numbers with very limited development of resistance wheneach bacteriophage is tested in isolation. However, when a mixture ofall four bacteriophages (Sa87, J-Sa36, Sa83, J-Sa37) is used,development of resistant forms is more rapid than when thebacteriophages are used in isolation, indicating antagonistic effects inthe mixed bacteriophage infection that permit enhanced bacterial escape.

Further testing clarifies that bacteriophage J-Sa37 appears to beantagonistic to the effects of bacteriophages Sa87, J-Sa36 and/or Sa83in reducing the development of bacterial resistance (as is measured byoptical density at OD600) illustrating the antagonistic effect ofJ-Sa37. An improved bacteriophage composition is provided havingbacteriophages Sa87, J-Sa36 and Sa83 which demonstrates enhancedtherapeutic efficacy when tested using the pneumonia model (see Examples3-5).

Example 7 Preparation of Mutants of Sa87, J-Sa36 and Sa83

Bacteriophages Sa87, J-Sa36, and Sa83 are iteratively passaged with S.aureus strains using conventional techniques (see for example Kelly etal, (2011), Bioengineered Bugs, 2:1, 31-37, which is incorporated hereinby reference). Escape phages capable of lysing S. aureus strainspreviously resistant are selected. Genetic mutation of said escapephages compared to bacteriophages Sa87, J-Sa36, and/or Sa83 is confirmedby genetic sequencing. The escape phages are selected for inclusion in abacteriophage composition comprising Sa87, J-Sa36, and/or Sa83 based onthe methodology of Example 6. The bacteriophage composition demonstratessimilarly enhanced therapeutic efficacy to the composition havingbacteriophages Sa87, J-Sa36, and Sa83 when tested using the pneumoniamodel (see Examples 3-5).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

What is claimed:
 1. A bacteriophage composition comprising one or morebacteriophages selected from Sa87, J-Sa36, and Sa83.
 2. Thebacteriophage composition of claim 1, comprising at least twobacteriophages selected from Sa87, J-Sa36, and Sa83.
 3. Thebacteriophage composition of claim 1, comprising purified bacteriophagesSa87, J-Sa36, and Sa83.
 4. A bacteriophage composition consistingessentially of Sa87, J-Sa36, and Sa83.
 5. The bacteriophage compositionof claim 1, further comprising an antibiotic.
 6. The bacteriophagecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier, diluent, excipient or combinations thereof.
 7. Thebacteriophage composition of claim 3, wherein the composition comprises1×10¹¹ PFU/mL per phage.
 8. The bacteriophage composition of claim 7,wherein the composition has a final endotoxin level less than 1000EU/mL.
 9. The composition of claim 8, wherein the composition is diluted1:10.
 10. The composition of claim 8, wherein the composition is diluted1:100.
 11. The composition of claim 1, wherein the composition isadministered to a mammal in a dosage range between about 1×10⁷ to about1×10¹¹ PFU of each phage at least once daily.
 12. A bacteriophagecomposition comprising two or more purified bacteriophage populationsselected from Sa87, J-Sa36, and Sa83.
 13. The bacteriophage compositionof claim 12, further comprising an antibiotic.
 14. The bacteriophagecomposition of claim 12, further comprising a pharmaceuticallyacceptable carrier, diluent, excipient or combinations thereof.
 15. Thebacteriophage composition of claim 12, wherein the composition comprises1×10¹¹ PFU/mL per phage.
 16. The bacteriophage composition of claim 15,wherein the composition has a final endotoxin level less than 1000EU/mL.
 17. The composition of claim 16, wherein the composition isdiluted 1:10.
 18. The composition of claim 16, wherein the compositionis diluted 1:100.
 19. The composition of claim 16, wherein thecomposition is administered to a mammal in a dosage range between about1×10⁷ to about 1×10¹¹ PFU of each phage at least once daily.
 20. A kitcomprising: a. a bacteriophage composition according to claim 1; and b.instructions for use of same.
 21. The kit of claim 20, furthercomprising an antibiotic and instructions for use of same in combinationwith the bacteriophage composition.
 22. The kit of claim 21, whereinsaid instructions are for use of same in treating a Staphylococcusaureus infection, preferably a Staphylococcus aureus pulmonaryinfection.